This study has been undertaken to investigate the mechanism(s) of mitogen mediated signal transduction induced by epidermal growth factor (EGF) and its receptor (EGFR) in primary cultured hepatocytes from normal and transgenic mice bearing albumin promoter/c-myc fusion genes in an attempt to define the involvement of c-myc in hepatic growth and oncogenesis. Insulin-stimulated, serum starved, cultured hepatocytes isolated from adult male B6CBAF-1/J normal and c-myc transgenic mice, were metabolically prelabeled with P-32 orthophosphate and then treated with EGF. The ad- ministration of EGF (5 min) to primary hepatocyte cultures resulted in phosphorylation of a number of substrates. The putative 170 kDa EGFR was immunoprecipitated with anti-phosphotyrosine (p-Tyr) antibody and was phosphorylated by EGF in a dose-dependent fashion. Two-dimensional poly- acrylamide gel electrophoresis (2D-PAGE) of total cellular lystate polypeptides from either normal or c-myc transgenic hepatocytes, either in the absence or in response to EGF treatment, resulted in complex 2D-maps consisting of 600-800 individual phosphoproteins. 2D-PAGE in combination with anti-phosphotyrosine (anti-pTyr) immunoprecipitation of whole cell lysates enabled the detection of significant differences in cellular phosphorylation between EGF stimulated normal and c-myc transgenic hepatocytes. Significant (4- to 10-fold) increases in protein phosphorylation was noted in at least 16 polypeptides in M-r range of 30- 70 kDa and pI of 5.0-6.0 in c-myc hepatocytes as compared to normal. No differences in the constitutive protein phosphorylation were observed in normal and c-myc hepatocytes in the absence of EGF treatment. These results suggest that alterations of specific protein phosphorylation may be due to differences in cellular metabolism cooperated by overexpression of the c-myc proto-oncogene.